Structure of microbial communities in activated sludge: potential implications for assessing the biodegradability of chemicals.

Various methods used to assess the biodegradability of chemicals often employ activated sludge as an inoculum since chemicals that ultimately enter the environment are often discharged through wastewater. Differences in the structure and function of activated sludge microbial communities that may complicate interpretation of biodegradation tests could arise from differences in wastewater composition, wastewater treatment plant (WWTP) operation, or manipulations done after collection of the activated sludge. In this study, various methods were used to characterize the structure of microbial communities found in freshly collected activated sludge from WWTPs in Japan, Europe, and the United States, as well as sludge that had been continuously fed either sewage or a glucose-peptone mixture for several weeks after collection. Comparisons of biomass levels, whole-community substrate utilization (determined using Biolog GN and GP plates), and phospholipid fatty acid (PLFA) profiles indicated there were both geographical and temporal differences among freshly collected activated sludge samples. Moreover, marked shifts in the structure of activated sludge microbial communities occurred upon continuous cultivation in the laboratory for 5 weeks using a glucose-peptone feed. These shifts were evident from whole-community substrate utilization and PLFA profiles as well as differences in the profiles of 16S rDNA genes from numerically dominant populations obtained by denaturing gradient gel electrophoresis and terminal restriction fragment analyses. Further studies are needed to better define the variability within and between activated sludge from wastewater treatment plants and laboratory reactors and to assess the impact of such differences on the outcome of biodegradability tests.

Seasonal temperature declines do not decrease periphytic surfactant biodegradation or increase algal species sensitivity.

The effects of seasonally decreasing river water temperature on surfactant biodegradation and algal sensitivity are reviewed from four stream mesocosm studies conducted over a 5-year period. Seasonal temperatures ranged from 28 to 0 degree C over all studies and temperature declines were approximately 9 to 14 degrees C over the course of each individual study. Mesocosm periphyton were naturally colonized on tile substrata with in-flowing river water for a period of 3 to 8 weeks prior to the initiation of sampling. Streams were dosed for 8 to 11 weeks with microgram/L (ppb) quantities of the surfactants C12-alkyl sulfate (C12-AS), C45E2.17S-alkyl ethoxysulfate (AES), C25E6-alkyl ethoxylate (AE) or 0 to 13% final effluent during the sampling period. Mineralization of C12-AS and AE by periphyton in the dosed streams generally increased over the dosing period while mineralization remained approximately constant in the control streams. The results from the AE study occurred with an increase in periphyton heterotrophic respiration. Mineralization of AES increased over the dosing period in streams receiving the highest dose of AES and remained constant in streams receiving lower doses. All studies involving surfactant exposure demonstrated a positive correlation between surfactant concentration and mineralization during periods of seasonal temperature decline. Mineralization of AE by periphyton dosed with final effluent increased slightly over the testing period. Periphytic algal taxonomy and biovolume were evaluated during the AES study. Overall, these tests showed no increases in species sensitivity over the testing period. Taken collectively, these results indicate that there is no correlation between naturally decreasing seasonal temperatures and lower rates of surfactant mineralization or increased species sensitivity by naturally acclimated periphyton.

Stream periphytic biodegradation of the anionic surfactant C12-alkyl sulfate at environmentally relevant concentrations.

The effects of continuous exposure to C12-alkyl sulfate on a periphytic microbial community were determined in an 8-week stream mesocosm study. C12-alkyl sulfate concentrations ranged from environmentally relevant (< 10-20 micrograms/liter) to unrealistically high concentrations (> 1500 micrograms/liter). Endpoints evaluated included turnover rates, bacterial cell density, heterotrophic mixed amino acid uptake, and fatty acid profile evaluations. Predosed periphyton demonstrated a mean turnover rate for C12-alkyl sulfate of 0.08/hr. During the 8-week dosing period, a significant increase in mean turnover rates was observed in streams dosed with > or = 61 micrograms C12-alkyl sulfate/liter, despite a 10 degrees C drop in stream temperature. A significant correlation between turnover rate and C12-alkyl sulfate concentration was also observed. While bacterial cell density increased during the study, it was determined that the biodegradation acclimation to C12-alkyl sulfate was not biomass-specific. Likewise, bacterial activity generally increased over the study, but it did not correlate with either biodegradation or bacterial cell density. Lastly, phospholipid fatty acid profiles indicate that a shift in the microbial community occurred in the high-dose stream as opposed to the control stream. This study demonstrates that C12-alkyl sulfate is rapidly degraded and induces a biodegradative acclimation response at environmentally relevant concentrations.

Membrane fatty acids as phenotypic markers in the polyphasic taxonomy of methylotrophs within the Proteobacteria.

A polyphasic approach to bacterial taxonomy attempts to integrate phylogenetic relationships with phenotypic marker analysis. This study describes the application of membrane fatty acids as a phenotypic marker for methylotrophs. Detailed phospholipid, ester-linked fatty acid (PLFA) profiles are reported for 17 methylotrophic eubacterial strains. These profiles included verification of double bond positions and geometries, both critical features for this analysis. Multivariate cluster analysis was used to indicate groupings of these strains along with literature values of both methylotrophs and non-methylotrophs based on the PLFA phenotype. Like many phenotypic characteristics, PLFA profiles were influenced by environmental conditions. The instabilities displayed, however, were predictable from physiological studies including increased trans/cis and cyclopropyl/cis ratios. Cluster analysis of PLFA profiles generated by separate investigators with different culture conditions indicated reproducibility by strain and species. The PLFA phenotype relationships compare favourably with phylogenetic associations based on 16S rRNA data for methylotrophs and will continue to be a valuable phenotypic marker for Proteobacteria taxonomy.

Archaebacterial ether lipid diversity analyzed by supercritical fluid chromatography: integration with a bacterial lipid protocol.

A strategy has been developed for archaebacterial lipid analysis which provides three times the information to describe archaebacterial isolates and is compatible with simultaneous eubacterial/eukaryotic lipid analysis of environmental samples. Eubacterial and micro-eukaryotic biomass, community structure, and nutritional status have been routinely defined in environmental samples by lipid analysis. Lipid profiles are also useful in eubacterial identification and taxonomy. Polar lipid or whole cell ester-linked fatty acids are generally analyzed by gas chromatography-mass spectroscopy. Archaebacteria are characterized by their ether-linked membrane lipids. There is, however, less diversity in the side chains of archaebacterial membrane lipids as compared the eubacterial ester-linked membrane lipids. The information content of the archaebacterial lipid profile was increased by separately analyzing the polar lipid, glycolipid, and lipid-extracted residue fractions. Identification and quantification were performed by supercritical fluid chromatography. Results are presented for three species of methanogens and four thermoacidophile isolates, and compared with a literature review.

Comparison of three techniques for administering radiolabeled substrates to sediments for trophic studies: Incorporation by microbes.

Three principal methods have been used to administer substrates to sediments: injection, porewater replacement, and slurry. Here we assess how each of these techniques affects incorporation of radiolabels into macromolecules of marine sedimentary microbes. Eighty-five cores of intertidal sand were collected in a randomized-block, factorial design. One set of cores received(14)C-bicarbonate/(3)H-thymidine and was incubated in the light; another set received(14)C-acetate/(3)H-thymidine and was incubated in the dark. Following a 5-hour incubation, sediments were analyzed for incorporation of radiolabel into lipid fractions (neutral, glyco-, and polar) and DNA. The three methods of isotope administration were also applied to cores subsequently analyzed for polar lipid phosphates and phospholipid fatty-acid (PLFA) profiles. In general, incorporation was greatest when injections were made, consistent with the prediction that incorporation would decrease as specific activity of the radiolabeled substrate was diminished by dilution. The ratio of(14)C from acetate incorporated into polar and glycolipid fractions indicated that a significant disturbance accompanied the porewater and slurry techniques. Substantial amounts of(3)H were recovered in the neutral-lipid fraction, indicating that thymidine was catabolized by sedimentary microbes and tritiated products were incorporated by eukaryotes. There were no significant differences in PLFA profiles or estimates of microbial biomass among methods or controls. Incorporation of(3)H into DNA was similar with all combinations of methods and radiocarbon substrates.(14)C was extensively incorporated into DNA, indicating that photoautotrophs and heterotrophs utilized radiocarbon from bicarbonate and acetate, respectively, for de novo synthesis of DNA. Injection is suggested as the method of choice, as it presents more flexibility in its application than porewater replacement and disturbs the consortia of gradients in sediments to a significantly lesser degree than porewater replacement and slurry.

Effect of nutrient deprivation on lipid, carbohydrate, DNA, RNA, and protein levels in Vibrio cholerae.

The response of Vibrio cholerae to low nutrient levels was determined by measuring the concentrations of lipids, carbohydrates, DNA, RNA, and proteins over a 30-day starvation period. Ultrastructural integrity was observed by transmission electron microscopy. Total lipids and carbohydrates declined rapidly within the first 7 days, while DNA and protein exhibited a more constant decline over the 30 days of starvation. In contrast, RNA showed little decrease upon starvation. Although neutral lipids were lost, the percentage of neutral lipids did not decline as rapidly as the phospholipids. Detectable levels of poly-beta-hydroxybutyrate disappeared completely by 7 days. Carbohydrate profiles revealed the relative loss of the five-carbon sugar ribose and N-acetylglucosamine and a relative increase in the total six-carbon sugars, especially glucose. Morphologically, ribosomes appeared to exhibit no structural change, while inclusion bodies and mesosomelike structures disappeared completely, and cell wall and membrane integrity was lost. The data suggest that V. cholerae differs somewhat from other marine vibrios in its response to low nutrients but shares some characteristics in common with them. The data also suggest that certain lipids and carbohydrates may provide the endogenous energy sources needed for dormancy preparation and cell maintenance under nutrient starvation.

Phospholipid ester-linked fatty acid profile changes during nutrient deprivation of Vibrio cholerae: increases in the trans/cis ratio and proportions of cyclopropyl fatty acids.

The phospholipid ester-linked fatty acids of 0-day-, 7-day-, and 30-day-starved cultures of Vibrio cholerae were compared. Statistically significant trends were noted in the fatty acid profiles as the cells starved. The amount of the cis-monoenoic fatty acids declined (e.g., 16:1 omega 7c: 0 day, 39%; 7 day, 18%; 30 day, 11%). In contrast, the saturated fatty acids, the cyclopropyl derivatives of the cis-monoenoic fatty acids, and trans-monoenoic fatty acids increased during starvation. For instance, the amounts of 16:1 omega 7t were: 0 day, 1%; 7 day, 13%; 30 day, 17%; which increased the trans/cis ratio for 16:1 omega 7 from 0.02 (0 day) to 0.70 (7 day) to 1.56 (30 day). This may be due to the reported high turnover rates of cis-monoenoic fatty acids of membrane phospholipids and the availability of enzymes for the metabolism of these isomers. During starvation-induced phospholipid loss, the cis-monoenoic fatty acids would, therefore, be preferentially utilized. The ability to either synthesize trans-monoenoic acids (which are not easily metabolized by bacteria) or modify the more volatile cis-monoenoic acids to their cyclopropyl derivatives may be a survival mechanism which helps maintain a functional (although structurally altered) membrane during starvation-induced lipid utilization. In addition, a trans/cis fatty acid ratio significantly greater than that reported for most bacterial cultures and environmental samples (less than 0.1) may be used as a starvation or stress lipid index. Such a ratio could help determine the nutritional status of ultramicrobacteria and other reported dormant cells in natural aquatic environments.(ABSTRACT TRUNCATED AT 250 WORDS)

Fourier transform-infrared spectroscopic methods for microbial ecology: analysis of bacteria, bacteria-polymer mixtures and biofilms.

Fourier transform-infrared (FT-IR) spectroscopy has been used to rapidly and nondestructively analyze bacteria, bacteria-polymer mixtures, digester samples and microbial biofilms. Diffuse reflectance FT-IR (DRIFT) analysis of freeze-dried, powdered samples offered a means of obtaining structural information. The bacteria examined were divided into two groups. The first group was characterized by a dominant amide I band and the second group of organisms displayed an additional strong carbonyl stretch at approximately 1740 cm-1. The differences illustrated by the subtraction spectra obtained for microbes of the two groups suggest that FT-IR spectroscopy can be utilized to recognize differences in microbial community structure. Calculation of specific band ratios has enabled the composition of bacteria and extracellular or intracellular storage product polymer mixtures to be determined for bacteria-gum arabic (amide I/carbohydrate C-O approximately 1150 cm-1) and bacteria-poly-beta-hydroxybutyrate (amide I/carbonyl approximately 1740 cm-1). The key band ratios correlate with the compositions of the material and provide useful information for the application of FT-IR spectroscopy to environmental biofilm samples and for distinguishing bacteria grown under differing nutrient conditions. DRIFT spectra have been obtained for biofilms produced by Vibrio natriegens on stainless steel disks. Between 48 and 144 h, an increase in bands at approximately 1440 and 1090 cm-1 was seen in FT-IR spectra of the V. natriegens biofilm. DRIFT spectra of mixed culture effluents of anaerobic digesters show differences induced by shifts in input feedstocks. The use of flow-through attenuated total reflectance has permitted in situ real-time changes in biofilm formation to be monitored and provides a powerful tool for understanding the interactions within adherent microbial consortia.